Teleost contributions to the understanding of mycobacterial diseases.

Few pathogens have shaped human medicine as the mycobacteria. From understanding biological phenomena driving disease spread, to mechanisms of host-pathogen interactions and antibiotic resistance, the Mycobacterium genus continues to challenge and offer insights into the basis of health and disease. Teleost fish models of mycobacterial infections have progressed significantly over the past three decades, now supplying a range of unique tools and new opportunities to define the strategies employed by these Gram-positive bacteria to overcome host defenses, as well as those host antimicrobial pathways that can be used to limit its growth and spread. Herein, we take a comparative perspective and provide an update on the contributions of teleost models to our understanding of mycobacterial diseases.

Teleost antimicrobial peptide hepcidin contributes to host defense of goldfish (Carassius auratus L.) against Trypanosoma carassii.

Hepcidin is an antimicrobial peptide and an iron regulatory protein that prevents the release of excess iron in the blood. There is evidence suggesting that teleost hepcidin is a major player in antimicrobial defense against various bacteria species, but little is known regarding the effects of teleost hepcidin in protozoan parasitic infections. We examined the role of hepcidin during the course of infection of goldfish with Trypanosoma carassii. Quantitative real-time PCR was used to determine the expression of hepcidin in goldfish immune organs during the course of T. carassii infection. During the acute phase of the T. carassii infection, the mRNA levels of hepcidin were up-regulated in liver and kidney. In contrast, an up-regulation of hepcidin mRNA expression in spleen was observed during the chronic phase of the infection. Furthermore, a synthetic goldfish hepcidin peptide induced trypanosome lysis in vitro, and parasite surface disruption was confirmed by scanning electron microscopy (SEM) analysis. These results suggest that, in addition to well-characterized direct antibacterial activities, teleost hepcidin also exhibits trypanocidal activity.

Mechanisms of Fish Macrophage Antimicrobial Immunity.

Overcrowding conditions and temperatures shifts regularly manifest in large-scale infections of farmed fish, resulting in economic losses for the global aquaculture industries. Increased understanding of the functional mechanisms of fish antimicrobial host defenses is an important step forward in prevention of pathogen-induced morbidity and mortality in aquaculture setting. Like other vertebrates, macrophage-lineage cells are integral to fish immune responses and for this reason, much of the recent fish immunology research has focused on fish macrophage biology. These studies have revealed notable similarities as well as striking differences in the molecular strategies by which fish and higher vertebrates control their respective macrophage polarization and functionality. In this review, we address the current understanding of the biological mechanisms of teleost macrophage functional heterogeneity and immunity, focusing on the key cytokine regulators that control fish macrophage development and their antimicrobial armamentarium.

Recombinant IL-4/13A and IL-4/13B induce arginase activity and down-regulate nitric oxide response of primary goldfish (Carassius auratus L.) macrophages.

We report on the expression analysis and functional characterization of IL-4/13A and IL-4/13B in goldfish. Quantitative analysis indicated the highest expression in the heart, spleen, brain, and kidney, with comparable expression patterns for both IL-4/13A and IL-4/13B. The mRNA levels of IL-4/13A and IL-4/13B in the immune cells examined were highest in macrophage and monocytes. Assessment of spleen mRNA following infection with Trypanosoma carassii, a prominent protozoan pathogen of fish, revealed decrease in IL-4/13B and arginase expression 14 days post infection, followed by an increase in IL-4/13B and arginase-2 at 28 days post infection. Recombinant forms of IL-4/13A and IL-4/13B induced an increase in arginase activity in macrophages in a dose-dependent manner. Recombinant IL-4/13A and IL-4/13B also induced significant increase in mRNA levels of arginase -2 in macrophages at 6, 12, 18 and 24 h after treatment. Furthermore, treatment with both IL-4/13 recombinants interfered with the IFNγ-induced nitric oxide response of macrophages. Our results suggest a conserved role of IL-4/IL-13 in induction of alternative activation phenotype in teleost macrophages.

Neutrophil contributions to the induction and regulation of the acute inflammatory response in teleost fish.

Neutrophils are essential to the acute inflammatory response, where they serve as the first line of defense against infiltrating pathogens. We report that, on receiving the necessary signals, teleost (Carassius auratus) neutrophils leave the hematopoietic kidney, enter into the circulation, and dominate the initial influx of cells into a site of inflammation. Unlike mammals, teleost neutrophils represent <5% of circulating leukocytes during periods of homeostasis. However, this increases to nearly 50% immediately after intraperitoneal challenge with zymosan, identifying a period of neutrophilia that precedes the peak influx of neutrophils into the challenge site at 18 h after injection). We demonstrate that neutrophils at the site of inflammation alter their phenotype throughout the acute inflammatory response, and contribute to both the induction and the resolution of inflammation. However, neutrophils isolated during the proinflammatory phase (18 h after injection) produced robust respiratory burst responses, released inflammation-associated leukotriene B(4), and induced macrophages to increase reactive oxygen species production. In contrast, neutrophils isolated at 48 h after infection (proresolving phase) displayed low levels of reactive oxygen species, released the proresolving lipid mediator lipoxin A(4), and downregulated reactive oxygen species production in macrophages before the initiation of apoptosis. Lipoxin A(4) was a significant contributor to the uptake of apoptotic cells by teleost macrophages and also played a role, at least in part, in the downregulation of macrophage reactive oxygen species production. Our results highlight the contributions of neutrophils to both the promotion and the regulation of teleost fish inflammation and provide added context for the evolution of this hematopoietic lineage.

Biology of Bony Fish Macrophages.

Macrophages are found across all vertebrate species, reside in virtually all animal tissues, and play critical roles in host protection and homeostasis. Various mechanisms determine and regulate the highly plastic functional phenotypes of macrophages, including antimicrobial host defenses (pro-inflammatory, M1-type), and resolution and repair functions (anti-inflammatory/regulatory, M2-type). The study of inflammatory macrophages in immune defense of teleosts has garnered much attention, and antimicrobial mechanisms of these cells have been extensively studied in various fish models. Intriguingly, both similarities and differences have been documented for the regulation of lower vertebrate macrophage antimicrobial defenses, as compared to what has been described in mammals. Advances in our understanding of the teleost macrophage M2 phenotypes likewise suggest functional conservation through similar and distinct regulatory strategies, compared to their mammalian counterparts. In this review, we discuss the current understanding of the molecular mechanisms governing teleost macrophage functional heterogeneity, including monopoetic development, classical macrophage inflammatory and antimicrobial responses as well as alternative macrophage polarization towards tissues repair and resolution of inflammation.

Development of an in vitro model system to study the interactions between Mycobacterium marinum and teleost neutrophils.

The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1ß1, il-1ß2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates.

Identification and functional characterization of the goldfish (Carassius auratus L.) high mobility group box 1 (HMGB1) chromatin-binding protein.

We report on the identification and functional characterization of HMGB1 of the goldfish. Quantitative analysis indicated the highest expression of goldfish HMGB1 in the brain, with lower mRNA levels in spleen, intestine, kidney, gill and heart. HMGB1 was also differentially expressed in goldfish immune cell populations with highest mRNA levels present in splenocytes and neutrophils. We generated and functionally characterized the recombinant HMGB1 (rgHMGB1). The rgHMGB1 primed the respiratory burst response in monocytes and induced nitric oxide production of primary goldfish macrophages. Treatment of goldfish macrophages with heat-killed Mycobacterium marinum and Aeromonas salmonicida elevated the expression of HMGB1 and resulted in higher HMGB1 protein levels. The rgHMGB1 induced a dose-dependent production of TNFα-2 and IL-1ß1 of goldfish macrophages. Furthermore, the dual luciferase reporter assay revealed that goldfish HMGB1 induced the activation of the NF-κB signaling pathway. Our results indicate that goldfish HMGB1 is a critical regulatory cytokine of inflammatory and antimicrobial response of the goldfish.

Antimicrobial responses of teleost phagocytes and innate immune evasion strategies of intracellular bacteria.

During infection, macrophage lineage cells eliminate infiltrating pathogens through a battery of antimicrobial responses, where the efficacy of these innate immune responses is pivotal to immunological outcomes. Not surprisingly, many intracellular pathogens have evolved mechanisms to overcome macrophage defenses, using these immune cells as residences and dissemination strategies. With pathogenic infections causing increasing detriments to both aquacultural and wild fish populations, it is imperative to garner greater understanding of fish phagocyte antimicrobial responses and the mechanisms by which aquatic pathogens are able to overcome these teleost macrophage barriers. Insights into the regulation of macrophage immunity of bony fish species will lend to the development of more effective aquacultural prophylaxis as well as broadening our understanding of the evolution of these immune processes. Accordingly, this review focuses on recent advances in the understanding of teleost macrophage antimicrobial responses and the strategies by which intracellular fish pathogens are able to avoid being killed by phagocytes, with a focus on Mycobacterium marinum.

Characterization of three Nod-like receptors and their role in antimicrobial responses of goldfish (Carassius auratus L.) macrophages to Aeromonas salmonicida and Mycobacterium marinum.

The nucleotide-binding oligomerization domain proteins Nod1, Nod2 and Nlrx1 are cytoplasmic pathogen recognition receptors (PRRs) of the Nod-like receptor (NLR) family. In this report, goldfish Nod1 (gfNod1), Nod2 (gfNod2) and Nlrx1 (gfNlrx1) genes were cloned and characterized. The full length of gfNod1, gfNod2 and gfNlrx1 were 3234bp, 3129bp and 4900bp, encoding 937, 982 and 1008 amino acids, respectively. The three Nod-like receptors have a NACHT domain and C-terminal leucine rich repeat (LRR) domains. In addition to these, gfNod1 and gfNod2 also had an N-terminal CARD domain (two in gfNod2). Phylogenetic analysis showed that the three NLRs are highly conserved. Quantitative gene expression analysis of the three receptors revealed the greatest mRNA levels in the spleen, and in isolated neutrophils and splenocytes. Furthermore, treatment of goldfish macrophages with LPS, Poly I:C, MDP, PGN, heat-killed Aeromonas salmonicida or Mycobacterium marinum differentially altered the expression of the Nod-like receptors. Our results indicate that Nod-like receptors are functionally highly conserved and that they play a pivotal role in recognition of fish pathogens such as A. salmonicida and M. marinum.

Analysis of the immune response in infections of the goldfish (Carassius auratus L.) with Mycobacterium marinum.

The rapid doubling time and genetic relatedness of the fish pathogen Mycobacterium marinum to Mycobacterium tuberculosis has rendered the former an attractive model for investigating mycobacterial host-pathogen interactions. We employed the M. marinum-goldfish infection model to investigate the in vivo immune responses to this pathogen in the context of a natural host. Histological analysis revealed mycobacterial infiltrates in goldfish kidney and spleen tissues, peaking 28 days post infections (dpi). Quantitative gene expression analysis showed significant increases of mRNA levels of pro-inflammatory cytokines (IFNγ, IL-12p40, IL-1ß1) and cytokine receptors (IFNGR1-1, TNFR2) at 7 dpi. Conversely, the gene expression levels of key anti-inflammatory cytokines TGFß and IL-10 were elevated at 14 dpi. Furthermore, M. marinum infections markedly increased the cytokine-primed oxidative burst responses of isolated kidney phagocytes at 7 but not 56 dpi. We believe that the M. marinum-goldfish infection model will be invaluable in furthering the understanding of the mycobacterium host-pathogen interface.

Analysis of the antimicrobial responses of primary phagocytes of the goldfish (Carassius auratus L.) against Mycobacterium marinum.

The slow growth rate of Mycobacterium spp. that infect humans coupled with a lack of reliable in vitro infection model systems has hindered the progress of research in host cell-mycobacteria interactions. Recent studies have utilized the relatively fast growing Mycobacterium marinum to examine the host-pathogen interface in natural fish hosts. Here we describe the use of primary goldfish monocyte and mature macrophage cultures to investigate the immune cell-M. marinum interactions. Live and heat-killed M. marinum abrogated the recombinant goldfish (rg)TNFα2 and rgIFNγ-induced monocyte reactive oxygen production. Live but not heat-killed M. marinum also ablated rgIFNγrel and rg-TNFα2 induced macrophage nitric oxide production. M. marinum induced significant changes in gene expression of select NADPH oxidase components and inflammatory cytokine receptors and up-regulated the expression of immunosuppressive genes IL-10, TGFß1 and SOCS-3. The exposure of monocytes and mature macrophages to M. marinum caused an increase in the mRNA levels of several pro-inflammatory genes. Stimulation of monocytes and macrophages with rgTNFα2, rgIFNγ, or rgIFNγrel reduced the survival of intracellular mycobacteria. The characterization of the interaction between M. marinum and natural host-derived primary phagocyte cultures will enable future studies on the host-pathogen interactions in mycobacterial infections.

Characterization and functional analysis of goldfish (Carassius auratus L.) interleukin-10.

This is the first report of comprehensive functional analysis of an interleukin-10 in bony fish. Quantitative expression analysis of goldfish IL-10 revealed the greatest mRNA levels in the spleen tissues, peripheral blood leukocytes and granulocytes. The stimulation of cells with recombinant goldfish (rg) TNFα2 significantly reduced IL-10 mRNA levels in granulocytes and monocytes of the goldfish. To functionally assess the goldfish IL-10, we generated a recombinant form of the molecule (rgIL-10). The rgIL-10 substantially reduced the expression of TNFα1, TNFα2, IL-1ß1, IL-10, CXCL-8, and NADPH oxidase component, p47(phox) in monocytes activated with heat-killed Aeromonas salmonicida and reduced the expression of IFNγ in A. salmonicida-activated splenocytes. Pre-treatment of monocytes with rgIL-10 resulted in substantial reduction of the ROI response of the A. salmonicida or rgIFNγ-primed monocytes. The rgIL-10 bound to goldfish monocytes and induced phosphorylation and nuclear translocation of Stat3. The rgIL-10 also induced rapid and robust increase in the mRNA levels of the goldfish monocyte SOCS-3. Our results indicate that the function of IL-10 is highly conserved through evolution.